The effects of locally delivered insulin on equine hoof lamellar tissue

Castro, Matias (2015). The effects of locally delivered insulin on equine hoof lamellar tissue MPhil Thesis, School of Veterinary Science, The University of Queensland. doi:10.14264/uql.2015.873

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Author Castro, Matias
Thesis Title The effects of locally delivered insulin on equine hoof lamellar tissue
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
DOI 10.14264/uql.2015.873
Publication date 2015-08-31
Thesis type MPhil Thesis
Supervisor Andrew van Eps
Christopher Pollitt
Martin Sillence
Total pages 137
Language eng
Subjects 0707 Veterinary Sciences
070706 Veterinary Medicine
07 Agricultural and Veterinary Sciences
Formatted abstract
The equine hoof lamellar tissue is a highly specialized structure that plays a critical role in the suspensory apparatus of the distal phalanx (SADP). Failure of this normal attachment between distal phalanx and hoof is a major consequence of laminitis. Several conditions have been associated with laminitis in the past, but those related to insulin resistance (IR) or excess of glucocorticoids appear to be involved in the majority of the reported cases. The common factor in these conditions is hyperinsulinemia. Besides the main role of insulin in regulating glucose metabolism, it is involved in diverse physiologic process such as vascular modulation and cellular growth and proliferation. The role of insulin in laminitis has been assessed by different authors and new experimental model has arisen recently to induce laminitis after systemic administration of high concentrations of the hormone. However, the exact mechanism involved in the pathophysiology of the disease remains unclear.

Utilizing the minimally invasive technique of tissue microdialysis, this work aimed to evaluate the local effects of insulin, delivered in higher than normal concentrations into the lamellar interstitial fluid, by assessing the suggested potential mechanisms involved in this form of the disease: vasodilation, derangements of energy metabolism, and epidermal cellular proliferation. The latter was assessed using 5-ethynyl-2’-deoxyuridine (EdU), a novel marker of mitotic activity.

Insulin was delivered locally to the forefeet of healthy, Standardbred horses using one of the limbs as treatment (n=6) and the contralateral as control (n=6). Although they did not reach significance, changes in lamellar glucose metabolism (decreased interstitial glucose) were observed after delivering an approximated amount of 0.5 µg/ml for 24h at 1 µL/min via microdialysis to the sublamellar tissue. No variations were detected in the other measured metabolites (lactate, pyruvate and urea). The local delivery of insulin did not affect the microcirculation of the lamellae (no changes in urea clearance). These results support the hypothesis of insulin modifying lamellar cellular metabolism by increasing glucose uptake and consumption. This metabolic shift may be relevant in terms of modifying the stability of the cytoskeleton of the epidermal basal cells (EBCs) and the organization of lamellar tissue.  

To determine if high concentrations of insulin delivered locally affected the proliferation rate of the EBCs, a second experiment was performed in eight healthy, Standardbred horses. The microdialysis protocol was completed as for the six initial horses. After insulin was delivered for 24h, the proliferation marker EdU (5-ethynil-2’-deoxyuridine) was administered to both forefeet (control and treatment). The EdU technique was developed and standardized as part of this project for the study of the lamellar proliferation. Significative changes in the proliferation rate or total number of cells were found after 24h of local delivery of high concentrations of insulin to the lamellar tissue. These changes were restricted to the tissue sections harvested from close to the microdialysis membrane (L1) of the treatment group. A histomorphometric analysis was added to the experimental protocol to examine changes in the organization of the lamellae. The primary epidermal lamellae (PELs) and the non-keratinized tip of the PELs (NKPELL) measured significantly longer in the tissue sections of the treatment group containing the microdialysis probe. No changes were detected in the length of the secondary epidermal lamellae (SELs) or the keratinized axis of the PELs (KPELL). These results suggest a possible direct effect of insulin on lamellar tissue organization, which could be responsible for weakening of the SADP as was hypothesized by previous authors.

This project has contributed to the understanding of the mechanism involved in the pathophysiology of insulin-induced laminitis. Despite the results being not completely conclusive, this work is the first to assess the direct local effect of insulin in the lamellar tissue and provides new questions and directions for future research. Further work should be focused on the study of the concentration and transport of insulin in the lamellar tissue as well as the interactions between insulin and its receptors in the lamellae. The study of the relationship between hyperinsulinemia, the cytoskeleton of EBCs and the failure of the SADP will contribute to the understanding of the insulin-induced laminitis model.
Keyword Equine laminitis
Cellular metabolism
Energy metabolism
Cellular proliferation
Urea clearance
Hoof growth

Document type: Thesis
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Created: Mon, 10 Aug 2015, 13:46:04 EST by Matias Castro on behalf of University of Queensland Graduate School